Known C-terminal sequencing methodologies are enzymatic physical or chemical. The enzymatic approach is basically a time-course carboxypeptidase procedure. It is limited by differential hydrolysis rates of the involved peptide bonds and by potential unaccessibility of the COOH carboxyl terminus in proteins. The approach may yield the correct amino acids but in the wrong order and may not extend to more than three to five amino acids.
Physical approaches include mass spectrometry and nuclear magnetic resonance (NMR) and are most suitable for small peptides. Fast atom bombardment--Mass Spectrometry (FAB/MS) sensitivity for determining an entire peptide sequence is in the range of 1-10 nmol and is limited to expensive multisector instruments. Micromolar samples are required for NMR analysis.
Four chemical methods of some interest are known. In 1978 Parkam and Loudon reported a method in which the carboxyamido peptide derivative is treated with bis(1,1 trifluoroacetoxy)iodobenzene to yield a derivative of the amino acid. Free COOH groups were treated with bis-p-nitrophenylphosphoryl azide to generate the carboxyamido derivative through a Curtius rearrangement..sup.1 FNT .sup.1 Parham, M.E. and Loudon G.M. Biochem. Biophys. Res. Commun. 80:1;7 (1978).
Loudon and coworkers presented another version of the method which entailed reaction of the COOH terminus with pivaloylhydroxyl amine in the presence of carbodimide to effect a Lossen rearrangement. This method failed to degrade aspartic and glutamine residues..sup.2 FNT .sup.2 Miller, m.J and Loudon, G.M., J. Am. Chem. Soc. 97:5296 (1975); Miller, M.J., et al., J. Org. Chem. 42:1750 (1977).
The method reported by Stark.sup.3 releases the COOH-terminal amino acid as a thiohydantoin. It entails activation of the COOH group with acetic anhydride, followed by reaction with ammonium thiocyanate and cleavage by acid or base hydrolysis to release the thiohydantoin from the peptide chain. FNT .sup.3 Stark, G.R. Biochemistry 8:4735 (1968); Stark, G.R. in "Methods in Enzymology", Vol. 25, p. 369, Academic Press, New York, N.Y. (1972).
Hawke reported a modification of the Stark chemistry in which trimethylsilylisothiocyanate is utilized as the coupling reagent..sup.4 FNT .sup.4 Hawke, et al. Analytical Biochemistry 166:298-307 (1987).
Notwithstanding these procedures, there is a continuing substantial need for a generally applicable chemical method for C-terminal sequencing. Such a method would have particular value with respect to, among other things, sequencing N-terminal blocked polypeptides and proteins, verifying the primary protein structures predicted from DNA sequences, providing practical detection of post translational processing of gene products from known codon sequences, and as an aid in the design of oligonucleotide cDNA or gene bank probes.